(Supported by the NIH/NCRR/P41 RR 01219 grant, NSF MCB9808879 to B. McEwen and NIH GMS 40198 to C. L. Rieder). Three decades of structural analyses have produced the view that the kinetochore in vertebrate cells is a disk-shaped structure composed of three distinct structural domains. The most prominent of these consists of a conspicuous electron opaque "outer plate" that is separated by a light staining electron translucent "middle" plate from an "inner" plate associated with the surface of the pericentric heterochromatin. Spindle microtubules terminate in the outer plate and, in their absence, a conspicuous "corona" of fine filaments radiates from the cytoplasmic surface of this plate. Here we report for the first time the ultrastructure of kinetochores in untreated and colcemid-treated vertebrate somatic (PtK1) cells prepared for optimal structural preservation using high-pressure freezing and freeze-substitution. In serial thin sections, and IVEM tomographic re construc tions, the kinetochore appears as a 50-75 nm thick mat of light-staining fibrous material that is directly connected with the more electron opaque surface of the centromeric heterochromatin. This mat corresponds to the outer plate in conventional preparations, and it is surrounded on its cytoplasmic surface by a conspicuous 100-150 nm wide zone that excludes all ribosomes and other cytoplasmic components. High magnification views of this zone reveal that it contains a loose network of light staining 9-nm thick fibers that are analogous to the corona fibers in conventional preparations. Unlike the chromosome arms which appear uniformly electron opaque, the chromatin in the primary constriction appears mottled. Since and electron-translucent "middle" plate is not visible in these kinetochore preparations this zone is likely an artifact produced during conventional fixation and/or dehydration procedures. McEwen, B.F., C-E.Hsieh, A. L. Mattheyses and C. L. Rieder. 1998. A new look at kinetochore structure in vertebrate somatic cells using high-pressure freezing and freeze substitution. Chromosoma (in press) McEwen, B.F., C-E. Hsieh, A. L. Mattheyses and C. L. Rieder. (1998). A new look at kinetochore structure in vertebrate somatic cells using high-pressure freezing and freeze substitution. Molecular Biology of the Cell